Silver nanoparticle toxicity on Artemia parthenogenetica nauplii hatched on axenic tryptic soy agar solid medium

The use of gnobiotic brine shrimp (Artemia spp.) for ecotoxicology and bacteria-host interaction studies is common. However, requirements for axenic culture and matrix effects of seawater media can be an obstacle. Thus, we investigated the hatching ability of Artemia cysts on a novel sterile Tryptic Soy Agar (TSA) medium. Herein, we demonstrate for the first time that Artemia cysts can hatch on a solid medium without liquid, which offers practical advantages. We further optimized the culture conditions for temperature and salinity and assessed this culture system for toxicity screening of silver nanoparticles (AgNPs) across multiple biological endpoints. Results revealed that maxima hatching (90%) of embryos occurred at 28 °C and without addition of sodium chloride. When capsulated cysts were cultured on TSA solid medium Artemia were negatively impacted by AgNPs at 30–50 mgL−1 in terms of the embryo hatching ratio (47–51%), umbrella- to nauplii-stage transformation ratio (54–57%), and a reduction in nauplii-stage growth (60–85% of normal body length). At 50–100 mgL−1 AgNPs and higher, evidence of damage to lysosomal storage was recorded. At 500 mgL−1 AgNPs, development of the eye was inhibited and locomotory behavior impeded. Our study reveals that this new hatching method has applications in ecotoxicology studies and provides an efficient means to control axenic requirements to produce gnotobiotic brine shrimp.

Nanoparticles of noble metals, such as silver, exhibit distinct physical, chemical, and biological properties 1 . Silver nanoparticles (AgNPs) are well known for their anti-bacterial, anti-viral, and anti-fungal activities 2 . AgNPs are therefore widely used as constituents within textiles, food packing, cosmetics, detergents, and paints, with further applications in biomedicine and water purification 3,4 . In the aquaculture industry, AgNPs are applied to improve water quality, as a functional feed additive, and for disease control 5 . However, the increasing application of AgNPs in consumer products and aquaculture leads to their inflow to aquatic environments which is of growing concern. The toxicity of AgNPs to numerous marine organisms has been reported, for example in brine shrimp (Artemia salina) 6,7 , white leg shrimp (Litopenaeus vannamei) 8 , the Mediterranean mussel (Mytilus galloprovincialis) 9 , copepods (Amphiascus tenuiremis) 10 , and rainbow trout (Oncorhynchus mykiss) 11 . The behaviour of AgNPs in seawater is complex due to their dissolution into free silver ions (Ag + ) and their variable aggregation level, which are influenced by the electrolyte composition of the media 12 . For example, AgNP toxicity to the marine medaka (Oryzia melastigma) is greater at higher salinities 13 , meanwhile, the bioaccumulation of AgNPs in clams (Scrobicularia plana) is greater at lower salinities 14 . AgNP toxicity also depends on their particle size and shape, concentration, and coating agent composition 15 . Different coating agents greatly influence the behavior, stability, and fate of AgNPs in various environments 16 . Hence, aqueous media such as seawater might be not ideal to study the toxicity of AgNPs due to many unknown interactions.
Another vital challenge in studying AgNP toxicity in vivo is the difficulty to eliminate unknown interactions between the host and microbial communities. Under conventional toxicology test conditions, the observed toxicity of AgNPs on brine shrimp (Artemia spp.) may in part reflect their influence on important host-associated microbial communities rather than solely through direct physiological impact on the host 17 . This problem can be addressed through the culture and use of gnotobiotic Artemia to eliminate complex effects of host-associated www.nature.com/scientificreports/ microbial communities on the host response to toxins, and to unravel important microbial influences 18 . However, contaminating sources of bacteria from the incubation seawater, air supply, and the cysts themselves require careful management.
To reduce microbial contamination, cysts must be decapsulated and disinfected with NaOH and NaOCl, neutralized with Na 2 S 2 O 3 , and washed with sterile seawater 19 . During incubation, the air supply must be filtered. Axenicity of Artemia nauplii are typically assessed on Marine Agar for five days, during which time toxicity screening has already begun or been completed. Although this protocol verifies the gnotobiotic condition of nauplii, when experiments are inadvertently initiated with non-sterile organisms prior to verification then inefficiencies are experienced since testing must be repeated 19 . This current culturing protocol is further disadvantaged since the effect of a tested compound on decapsulated cysts might also differ from that of intact cysts. Development of a more efficient method to produce gnotobiotic Artemia without a seawater medium and with a reduced risk of microbial contamination would thus offer a solution to these problems.
Tryptic Soy Agar (TSA) is a general-purpose medium for the isolation and cultivation of fastidious or nonfastidious microorganisms. Sterility testing for facility contamination typically requires an all-purpose medium such as TSA, which can support the growth of a broad range of bacteria and fungi 20 . Hence, TSA could potentially provide a novel solid medium for hatching Artemia whilst affording the added benefit of an in-situ monitoring system for microbial contamination. Without using seawater during incubation, the variation of AgNPs toxicity due to seawater's variable ionic and dissolved organic composition could further be reduced 21 .
Given the potential of TSA as an effective incubation media to produce gnotobiotic Artemia, testing different conditions (e.g., salinity, temperature) during hatching is importantfor optimization and standardization. Herein, we aim to investigate whether Artemia cysts can hatch on solid TSA media and explore the possibility of using this novel technique for AgNP toxicity testing.

Methods
Artemia hatching preparation. A. parthenogenetica cysts were harvested in the Aral Sea (Uzbekistan) in 2020 and frozen for 24 months. After transfer to the MTT Joint Stock company (Vietnam) they were washed with brine water in a 50 L conical tank, and cysts which sank were collected on a 120 µm mesh sieve and excess water was removed. The moist cysts were air-dried at 38-40 °C within 8 h to decrease their moisture content below 8%. These cysts were stored in ziplock bags at 4℃ until used. To prepare the axenic solid medium for hatching cysts, 40 g of Trypic Soy Agar (TSA) powder (Merck, Germany) was prepared in 1 L of either distilled water or sodium chloride solution according to the experimental design below. The mixture was shaken for 5 min and boiled for the TSA to completely dissolve then sterilized via autoclave (121 °C; 15 min). After cooling to 45-50 °C, silver nanoparticles at different concentrations were added. 15 mL of these TSA stocks were spread evenly covering the surface of a Petri plate. TSA Petri plates were covered with the upper lid and subjected to UV light (55 W; 15 min) to maintain axenic conditions (Fig. 1). 0.001 g of Artemia cysts were sprinkled evenly on the www.nature.com/scientificreports/ surface of each TSA media. TSA plates were re-covered with the upper lid and with aluminum foil to avoid light exposure. All hatching procedures were conducted in a biosafety cabinet to minimize any bacterial contamination. All equipment were disinfected with 70% alcohol in the biosafety cabinet and UV-treated for 40 min prior to cyst preparation. There were 10 Petri plate replicates for each treatment unit.
Silver nanoparticles. The AgNPs were obtained from Sil-Life™ (Taiwan) as a brown aqueous solution dissolved in pure water as the solvent. Properties of the AgNPs, as provided by the manufacturer, are displayed in Table 1.

Effect of temperature and salinity.
To test the effect of temperature on Artemia hatching success, the TSA Petri plates with cysts were incubated at four temperature levels: 22, 25, 28 and 31 °C. The effect of salinity was investigated using four concentrations of sodium chloride, which were incorporated during the preparation of the TSA Petri plates: 10, 20, 30, 40 gL −1 . Sodium chloride was dissolved in distilled water according to the treatment levels.
Effect of AgNPs. The salinity and temperature levels which showed the best hatching performance in the first two experiments were selected to test the effect of AgNPs, using eight AgNP nominal concentrations: 30, 50, 100, 500, 1000, 2000, 3000 and 5000 mgL −1 . A control was included without the addition of AgNPs.
Hatching performance and morphological analysis. In the first two experiments (temperature and salinity effect) only the umbrella ratio was determined while in the third experiment the umbrella and the nauplii ratios were assessed. Both parameters were determined at 24 h and 48 h post incubation. Umbrella-stage individuals were counted if the cyst had burst and the embryo had appeared. Nauplii were counted if the hatching membrane had ruptured and the free-swimming form was present.
(1) The percentage of umbrella-stage individuals: (2) The percentage of nauplii individual: The body length of ten random nauplii from each plate were measured under an inverted microscope (Nikon EL WD 0.3, Japan). The behavioral activity and morphology of those nauplii were also observed and described.

Lysosomal damage.
To determine lysosomal damage, the neutral red uptake (NRU) assay 22 was applied with modification. This assay is based on the binding ability of viable cells in the nauplii to the supravital dye neutral red (3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride) within lysosomes. Neutral red was dissolved in phosphate buffer solution (PBS) at 40 µg/mL then centrifuged at 600×g for 10 min to remove any precipitated crystals. The de-stain solution was prepared by mixing 50 mL ethanol (96%), 1 mL acetic acid and 49 mL deionized water. Ten nauplii at 48 h post incubation in the third experiment were pipetted into a 2 mL Eppendorf tube. 100 µL of the neutral red solution was added into each tube. The tubes were incubated for 2 h at 28 °C and then the dye solution was removed. Nauplii in each tube were washed with 150 µL PBS and then the  www.nature.com/scientificreports/ PBS was removed. Next, 150 µL of the de-stain solution was added into each tube and gently shaken for 10 min to minimize the low affinity binding between neutral red and any untargeted substance. Lastly, those nauplii were transferred to Petri plates and observed under an inverted fluorescent microscope (Ex 525 nm; Em 645 nm).

Statistical analysis.
Hatching and nauplii percentage data were arcsine-transformed prior to statistical analysis to stabilize variances and normalize proportional data. Transformed data of hatching and nauplii percentage, and body length among treatments were compared using one-way analysis of variance (ANOVA) with pair-wise Tukey's post hoc tests (α = 0.05) using SPSS software v.25.

Results
Effect of temperature. Temperature influenced the hatching percentage of Artemia cysts on the TSA plates. The hatching ratios 24 h post incubation at 25, 28 and 31 °C were significantly higher (p < 0.05) than that at 22 °C (Fig. 2). However, the hatching ratios among the three higher temperatures showed no significant difference (p > 0.05). There was a clear unimodal trend of hatching dependence on temperature at 48 h post incubation (Fig. 2). The mean hatching percentage increased from around 52% at 22 °C to 90% at 28 °C, then declined to 76% at 31 °C (p < 0.05).
Effect of salinity. The hatching ratios of Artemia cysts on the TSA plates were affected by the inclusion of sodium chloride in the media (Fig. 3). The hatching success 48 h post incubation decreased significantly (p < 0.05) from 90% in non-treated controls to around 60% at all levels of sodium chloride treatment. However, there was no difference in hatching ratios among sodium chloride treatments (p > 0.05). The same trend of hatching percentage was observed at 24 h post incubation.   (Figs. 4, 5). The hatching ratios at 24 h post incubation in the control and 30 mgL −1 AgNPs treatment were significantly higher (p < 0.05) than that in the other treatments (Fig. 4a). Interestingly, there was no difference in hatching ratios among 50-5000 mgL −1 AgNP treatments. A similar trend to 24 h post incubation observation was seen at 48 h post incubation. The hatching ratio in the control and 30 mgL −1 treatments achieved almost 90% and were higher than for other treatments (Fig. 4b).
In contrast, a stronger monotonically-based concentration effect of AgNPs was observed on nauplii stage transformation (p < 0.05) (Fig. 5). The nauplii percentage dropped from 80% in the control to 60% in the 30 and 50 mg L −1 treatments then to 27% in the 500 and 1000 mg L −1 treatments after 24 h (Fig. 5a). Although there was no difference in the nauplii ratio between the control and the 30 mg L −1 treatment after 48 h, there was a still clear trend of decreasing nauplii metamorphosis according to the increase of AgNPs inclusion (Fig. 5b). Noticeably, at ≥ 2000 mg L −1 AgNPs, nauplii were still absent after 48 h incubation. inclusion into the media. There was a decreasing trend in the BL with the increasing levels of AgNPs (Fig. 6). Control nauplii were the largest (BL ≈ 830 μm) while exposure to 1000 mgL −1 AgNP treatment resulted in substantial impact (BL ≈ 500 μm). The lowest AgNP dose tested (30 mgL −1 ) reduced (p < 0.05) nauplii BL to around 85% of their non-treated counterparts. Although BL was reduced, the appearance and behaviour of nauplii was otherwise normal up to 100 mgL −1 AgNP treatment (Fig. 7a-d). Those nauplii from the control, 30, 50, and 100 mgL −1 AgNP treatments were still actively swimming. Morphological and behavioural abnormalities (i.e., absence of eyes and weak movement) were observed at AgNP doses of 500 and 1000 mgL −1 (Fig. 7e,f). At higher AgNP concentrations no umbrella cysts succeeded in becoming nauplii (Fig. 7g-i).
Lysosomal damage. Although lysosomal damage was not quantitatively determined, there was a clear qualitative trend of increasing lysosome storage dysfunction with exposure of Artemia to increasing AgNP concentration (Fig. 8). In non-treated control animals, only a few bright white dots as lysosome were observed  www.nature.com/scientificreports/ (Fig. 8a). The proportion of white increased to create a large volume in the nauplii under higher levels of AgNP treatment (Fig. 8b-f); especially at 1000 mgL −1 AgNPs the abnormal nauplii were full of lysosome (Fig. 8f).

Discussion
The effect of temperature on the hatching of Artemia cysts on any solid medium (e.g., TSA) has not been reported to our knowledge. However, thermal effects on hatching and development of Artemia have substantially been studied in aqueous media. The highest hatching percentage of A. franciscana was reported at either 25 °C or 28 °C depending on the harvesting source 21,23 . Meanwhile, the optimum temperature for A. urmiana was 30 °C 24 . In contrast, the hatching percentage was reduced with the increasing temperature levels from 25 to 30 °C for Artemia cysts from Great Salt Lake, USA 25 . With the solid TSA medium in our study, 22 °C decreased hatching percentages of A. parthogenetica cysts compared to other temperature levels. However, after 48 h, the optimum temperature in our study (i.e., 28 °C) was similar to that in previous studies 21,26 . The effect of salinity on hatching Artemia cysts incubated on TSA solid medium also has not reported to our knowledge. Although, like for temperature, previous findings have determined the effect of salinity in aqueous media 26,27 . When testing with a wide range of salinity levels (15,45,60,75, and 90 ppt) no difference was found in the hatching percentage of cysts 27 . In contrast, when experimenting with a narrow range of salinity levels the optimum level was determined at 28 ppt 26 . Surprisingly, on the TSA solid medium which contained 5 gL −1 of sodium chloride, the hatching percentage was the highest in this study. Adding more sodium chloride in the TSA medium caused reduction in hatching success. These observations differ from results of previous studies using aqueous media.
AgNPs are known to be toxic to Artemia spp. Most studies thus far have utilized newly hatched first instar nauplii at test organisms. The 48 h EC 50 of AgNPs when exposed to hatched nauplii varies widely (ca. 20 to > 100 mgL −1 ) in interlaboratory studies based on the 'immobilization assay' [28][29][30] . However, some earlier studies indicate that when using hydrated cysts as the initial test life stage then sensitivity to metals may be increased. The hatching success of decapsulated A. franciscana cysts decreased from 74% (control) to 32% after 24 h exposure to 10 mgL −1 AgNPs 31 . Here, we applied early exposure of non-decapsulated Artemia to AgNPs for the first time and assessed multiple toxicity end points (i.e., hatching and metamorphosis success, growth, eye development, and behaviour).
Results reveal that AgNP exhibited initial toxic effects at 30-50 mgL −1 under our test conditions, although not as severely as the previous report 31 which may be due to capsulation and media effects. For example, apparent toxicity differences may stem from lower activation of Ag + in the TSA solid medium compared with seawater. Surprisingly, hatching success was similarly affected by AgNPs from 50 to 5000 mgL −1 . How cysts could resist www.nature.com/scientificreports/ such levels of AgNPs is uncertain. The cyst consists of five layers: the cuticular layer, alveolar layer, outer cuticular membrane, fibrous layer, and the inner cuticular membrane 32 . These layers serve as a filter to defend against environmental stressors and conserve the cell during the incubation process 33,34 . During the hydration process of the dried cyst, the volume of the embryo can increase to a maximum of 140% water content 35 , providing a direct route for AgNPs/Ag + into the embryo. Active metabolism initiates when cysts achieve around 60% water content and beyond 34 and might be affected by AgNPs/Ag + uptake during this time. However, after dried cysts completely change their form from biconcave to spherical, they cannot not absorb any more water, and presumably AgNPs/Ag + , which may play a part in why hatching success did not change among the 50-5000 mgL −1 AgNPs treatments. However, after 24 h there were no nauplii in the 2000-5000 mgL −1 AgNPs treatments. It is suggested that AgNPs and/or Ag + could penetrate through the epidermis layer of the umbrella cysts and interfere with embryo development. Hence, AgNPs affected the artemia nauplii more severely than the cyst. Further work is needed to isolate the effect of Ag + , AgNPs, and coating substances. This study showed that AgNPs cause Artemia nauplii abnormalities when incubated on solid media. Body length and the nauplii metamorphosis ratio were impacted at low AgNPs concentrations while the eye was completely under-developed at high AgNPs concentrations. When the nauplii were exposed to AgNPS in seawater abnormal nauplii were also observed 6 . Similar morphological changes such as deformation or developmental retardation of the eye, shrinking of the intestinal gut tract, degradation of the outer shell, as well as body length reduction were observed in A. salina nauplii exposed to ZnO NPs and TiO 2 NPs 36 . Such nanoparticles might affect eye development in arthropods by disrupting endocrine-mediated processes and inhibiting ecdysteroid biosynthesis, similar to the mechanism of some organic pollutants such as bisphenol and sodium decocyl sulfate 37 .
Damage to lysosome storage was observed in nauplii even without obvious abnormal morphology after being exposed to lower AgNP levels and was extreme at higher doses. By increasing the intra-lysosomal pH, AgNPs interfered with lysosomal enzyme activity 38 . The increase of lysosomal storage may represent a compensation mechanism for impaired lysosome function caused by nanoparticles 39 . Lysosomal destabilization was also observed in AgNP-exposed hepatopancreas cells of oysters (Crassostrea virginica) 40 . Similarly, the number of lysosomes in the hepatocytes of rainbow trout (Oncorhynchus mykiss) exposed to AgNPs increased 41 . Lysosomes are known as vital intracellular organelles which are indicators for the cytotoxicity of nanomaterials 42 , hence, the dysfunction of lysosomal storage can be used for further toxicity assessment research in Artemia nauplii.
Artemia spp. are used extensively as test organisms in toxicology studies, however, for the shrimp to become an officially international recognized biological model in nanoecotoxicology further efforts are necessary 30 . Since Artemia cysts are typically incubated in liquid medium they are likely affected by variable aeration conditions, pH, seawater ion compositions, and microbial communities. For example, the toxicity results of heavy metals on the hatching success of Artemia was affected by Ca 2+ and HCO 3− concentrations in the media 43,44 . Hence, evaluation of hatching success is a difficult toxicity test to standardize with issues in reliability, reproducibility, and compatibility of toxicity data 45 . Our results reveal that a TSA solid medium is an applicable incubation technique which benefits from having a chemical composition that is easily controlled. Bioavailability of toxins within this medium is an area which requires further evaluation as this is currently unascertained. We envision further applications of this technique to facilitate the study of host-microbe interactions, mechanisms of innate immunity, nutritional requirements, and metabolic functions in Artemia 17 .

Conclusion
Conventional methods to produce gnotobiotic Artemia in liquid media have practical limitations. We developed a novel solid media technique to hatch and culture Artemia which benefits from being more efficient and controllable than previous protocols. Cysts can hatch on TSA solid medium whilst exhibiting the axenic condition during the experimental period, and this technique also benefits from being able to monitor potential microbial contamination in-situ. Toxic effects of silver nanoparticles were evaluated on this medium to demonstrate applicability for ecotoxicological-based research, with further applications in host-microbe interaction studies being envisioned. Additional types of solid medium could be tested in future studies, and, although we have demonstrated proof of concept in this research, direct comparisons of liquid versus solid mediums in toxicity testing is advised.

Data availability
The data that support the findings of this study are available from the corresponding author, [LVD], upon reasonable request.